Abstract Innate immunity is an ancient defense response that evolved with the earliest metazoan creatures, and is the first line of defense against microbial infection. These responses rely on the immediate recognition of microbes by germline-encoded receptors, and drive the production of numerous chemical, biological, and cellular responses to defend against infection. In the face of constant microbial assault, innate immunity is essential for the survival of nearly all multicellular organisms. On the other hand, over-exuberant or inappropriate innate immune responses are the underlying cause of morbidity and mortality associated with many infectious, autoimmune, and autoinflammatory diseases. Thus, a thorough mechanistic understanding of innate immunity has many potential applications in the development of the next generation of therapeutics. This proposal uses the fruit fly Drosophila melanogaster as a model for the study of innate immunity. Flies offer many advantages for the study of innate immunity, including experimental tractability and a model system without the complexity of the adaptive immune response. The Drosophila immune response is an excellent model for vector insect species, and discoveries made in flies are being translated into new approaches to control vector-borne diseases. Furthermore, many aspects of the innate immune responses are highly conserved with mammals, and discoveries made in flies can be translated into important, paradigm shifting, findings in mammals. Particularly relevant for this proposal is the conserved NF-?B signaling pathway that drives the immediate response to infection, in both insects and mammals. In Drosophila, systemic microbial infections are recognized by two distinct NF-?B signaling pathways, the Toll and immune deficiency (Imd) pathways. Both of these pathways are triggered by microbial cell walls and drive the production of antimicrobial peptides and other immuno-protective molecules. In particular, the Imd pathway is triggered by DAP-type peptidoglycan from the cell wall of certain bacteria. The long-term objective of this proposal is to understand in molecular detail the mechanisms used by the Imd pathway to trigger effective immune responses. The specific aims of this proposal address the molecular mechanisms involved in peptidoglycan recognition and Imd signal transduction. Aim1 probes the function of amyloid fibrils formed by both the peptidoglycan receptors and key adapter proteins in this pathway. Aim 2 is focused on the ubiquitin dynamics that temporally regulate the Imd pathway, involving both activating K63-chains and response-limiting K48-polyubiquitination. Aim 3 involves the characterization of a new family of transporters involved in delivering peptidoglycan fragments, e.g. muramyl-dipeptide and tracheal cytotoxin, to cytosolic innate immune receptors, in flies and mammals.